Fig 5.

Enhancement of RSV frameshifting by combinatorial changes. (A) Mutations in pMV-RCAS are shown, with pseudoknot mutations color coded as described for Fig. 2A. The wild-type slippery sequence is underlined, and variants with slippery sequences UUUAAAC (IBV), AAAUUUU (SRV), and AAAUUUAAAC (IBV2) are indicated. (B) Amino acid changes in Gag and Pol generated by slippery sequence mutations. The critical asparagine involved in protease dimerization is outlined in red. The amino acid sequence given for the IBV2 mutant is based on the assumption that the frameshift event occurs at the IBV slippery sequence (UUUAAAC). Frameshifting may also occur at the original RSV slippery sequence (AAAUUUA), which is maintained, and this would give an additional leucine at the N terminus of Pol. The amino acid changes in Pol lie in a seven-amino-acid spacer region thought to tolerate point mutations (45) (Fig. 2C). (C) Ribosomal frameshift assays of pMV-RCAS variants. mRNAs were prepared, translated, and analyzed as described for Fig. 2. (D) Comparison of ribosomal frameshifting efficiencies measured for pMV-RCAS-derived mRNAs translated at 30°C or 39°C.